Wortmannin-sensitive phosphorylation, translocation, and activation of PLCgamma1, but not PLCgamma2, in antigen-stimulated RBL-2H3 mast cells.
In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcepsilonRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcepsilonRI cross-linking activates both PLCgamma1 and PLCgamma2. Activation is accompanied by the increased phosphorylation of both PLCgamma isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCgamma isoforms have distinct subcellular localizations. PLCgamma1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCgamma1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCgamma2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcepsilonRI cross-linking. The activation of PLCgamma1, but not of PLCgamma2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCgamma1 with little inhibition of PLCgamma2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCgamma1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCgamma1. Although less abundant than PLCgamma2, activated PLCgamma1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.
Barker
S A
SA
Department of Pathology and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Caldwell
K K
KK
Pfeiffer
J R
JR
Wilson
B S
BS
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D000941:Antigens / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D004140:Dinitrophenols / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D015471:Leukemia, Basophilic, Acute; D008407:Mast Cells / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine / Q000032:analysis; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000032:analysis / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D000941:Antigens; D004140:Dinitrophenols; D004791:Enzyme Inhibitors; D015544:Inositol 1,4,5-Trisphosphate; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D010768:Phosphoserine; D019000:Phosphotyrosine; D017455:Receptors, IgE; D012710:Serum Albumin, Bovine; D010738:Type C Phospholipases; D000077191:Wortmannin; C034638:dinitrophenyl-bovine serum albumin